Hypoxia and Hypoxia-Inducible Factors in Normal and Malignant Breast Epithelium

Breast cancer treatment is based on tumor and patient related factors such as tumor stage, grade, hormonal status, HER2 status, patient age, and family history to name a few. It is today widely acknowledged that hypoxia and hypoxia-inducible factors (HIF:s) contribute to tumor progression. We therefore set out to explore the impact of hypoxia on mammary epithelial differentiation and what consequences the hypoxic response might have on breast cancer development and behavior. We found that hypoxia induces a less differentiated, estrogen receptor (ER)-negative/cytokeratin (CK) 19-positive phenotype in mammary cancer cells in vivo and in vitro and suggest that hypoxiainduced dedifferentiation is one of the mechanisms behind hypoxia-driven malignant progression. Hypoxia was also found to significantly impair both morphological and functional differentiation of non-transformed, immortalized epithelial mammary cells grown in three-dimensional cultures. Heterogeneous ER expression in breast cancer was additionally found to be related to cyclin D1, a cell cycle regulator frequently overexpressed in breast cancer. As cyclin D1 expression was not affected by hypoxia, our findings suggest two separate mechanisms behind varied ER expression in breast cancer. The two pivotal regulators of hypoxic response, HIF-1α and HIF-2α, were analyzed in two cohorts of breast cancer patients and found to be un-correlated suggesting HIF-α subtype specific mechanisms of induction. Furthermore, HIF-2α was found to be an independent prognostic factor related to distant recurrence

Hypoxic conditions induce a cancer-like phenotype in human breast epithelial cells.

INTRODUCTION: Solid tumors are less oxygenated than their tissue of origin. Low intra-tumor oxygen levels are associated with worse outcome, increased metastatic potential and immature phenotype in breast cancer. We have reported that tumor hypoxia correlates to low differentiation status in breast cancer. Less is known about effects of hypoxia on non-malignant cells. Here we address whether hypoxia influences the differentiation stage of non-malignant breast epithelial cells and potentially have bearing on early stages of tumorigenesis. METHODS: Normal human primary breast epithelial cells and immortalized non-malignant mammary epithelial MCF-10A cells were grown in a three-dimensional overlay culture on laminin-rich extracellular matrix for up to 21 days at normoxic or hypoxic conditions. Acinar morphogenesis and expression of markers of epithelial differentiation and cell polarization were analyzed by immunofluorescence, immunohistochemistry, qPCR and immunoblot. RESULTS: In large ductal carcinoma in situ patient-specimens, we find that epithelial cells with high HIF-1α levels and multiple cell layers away from the vasculature are immature compared to well-oxygenated cells. We show that hypoxic conditions impaired acinar morphogenesis of primary and immortalized breast epithelial cells grown ex vivo on laminin-rich matrix. Normoxic cultures formed polarized acini-like spheres with the anticipated distribution of marker proteins associated with mammary epithelial polarization e.g. α6-integrin, laminin 5 and Human Milk Fat Globule/MUC1. At hypoxia, cells were not polarized and the sub-cellular distribution pattern of the marker proteins rather resembled that reported in vivo in breast cancer. The hypoxic cells remained in a mitotic state, whereas proliferation ceased with acinar morphogenesis at normoxia. We found induced expression of the differentiation repressor ID1 in the undifferentiated hypoxic MCF-10A cell structures. Acinar morphogenesis was associated with global histone deacetylation whereas the hypoxic breast epithelial cells showed sustained global histone acetylation, which is generally associated with active transcription and an undifferentiated proliferative state

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Differential regulation as well as target gene specificity of the two hypoxia-inducible factor (HIF)-alpha subunits HIF-1alpha and HIF-2alpha in various tumors and cell lines have been suggested. In breast cancer, the prognostic significance of HIF-1alpha is not clear-cut and that of HIF-2alpha is largely unknown. Using IHC analyses of HIF-1alpha, HIF-2alpha, and vascular endothelial growth factor (VEGF) expression in a tissue microarray of invasive breast cancer specimens from 512 patients, we investigated the expression patterns of the 2 HIF-alpha subunits in relation to established clinicopathologic variables, VEGF expression, and survival. HIF-1alpha and HIF-2alpha protein levels and their effect on survival were additionally analyzed in a second cohort of 179 patients. To evaluate the individual role of each subunit in the hypoxic response and induction of VEGF, HIF-alpha protein and HIF-alpha and VEGF mRNA levels were further studied in cultured breast cancer cells after hypoxic induction and/or knockdown of HIF-alpha subunits by siRNA by Western blot and Quantitative Real-Time PCR techniques. We showed that although HIF-1alpha and HIF-2alpha protein levels in breast cancer specimens were not interrelated, high levels of both HIF-1alpha and HIF-2alpha associated to high VEGF expression. HIF-2alpha expression was an independent prognostic factor associated to reduced recurrence-free and breast cancer-specific survival, whereas HIF-1alpha did not exhibit these correlations. In cultured cells, acute hypoxia induced both HIF-proteins. At prolonged hypoxia, HIF-2alpha remained accumulated, whereas HIF-1alpha protein levels decreased, in agreement with the oxygen level and time-dependent induction of HIFs recently reported in neuroblastoma

ID1 expression and histone acetylation in breast epithelial cells in normoxic compared to hypoxic 3D-cultures.

<p><b>A.</b> Relative mRNA expression of the transcription modulating ID factors, <i>ID1</i> and <i>ID2</i> in MCF-10A cells after 21 days of 3D-culture on ECM-derived substrate under normoxic and hypoxic conditions (left). Showing data from three independent experiments. Statistical analysis was performed with Student’s paired t-test (p). Immunohistochemical staining for ID1 on paraffin-embedded MCF-10A acini-like structures after 21 days of 3D-culture at normoxia (21%) or hypoxia (1%) (right). Size bars 20 µm. <b>B.</b> Acetylated histone 4 (AcH4) visualized by immunofluorescence (green) in normoxic (21%) and hypoxic (1%) human primary breast epithelial cell (left) and MCF-10A cell (right) acini-like structures at the indicated days post seeding. Actin was visualized by phalloidin staining (red). Size bars 20 µm. <b>C</b>. Percentage of MCF-10A cells in acini-like structures with global histone acetylation, i.e. positive for AcH4, 21 days post-seeding at normoxia (open boxes) and hypoxia (black boxes), showing data from three independent experiments. At least 200 cells were calculated in each experiment. Statistical analysis was performed by Student’s paired t-test (p). <b>D.</b> Immunoblot of AcH4 in MCF-10A cells in 3D-culture at 21% (left) and 1% (right) on ECM-derived substrate for 10 days. SDHA was used as a loading control. <b>E.</b> Non-malignant breast epithelial cells grown on differentiation-inducing ECM have an organized cell shape and a high degree of deacetylated histones; these cells differentiate and become post-mitotic. Contrary, breast epithelial cells grown as monolayer without ECM do not receive/accept signals to induce differentiation, leading to sustained global histone acetylation and opening of the chromatin for transcription, resulting in impaired differentiation and/or dedifferentiation accompanied with cell proliferation. Hypoxia, including hypoxic induction of ID1, promotes a proliferative and undifferentiated state in breast epithelial cells despite contact with the ECM.</p

Proliferation and cell death in hypoxic and normoxic 3D-cultures in ECM-derived substrate. A

<p>Ki-67 immunofluorescence (green) and actin (red) staining of primary human breast epithelial cells in 3D-culture in ECM-derived substrate at 21% and 1% oxygen for 12, and 21 days. Representative images from one of three independent experiments with breast epithelial cells isolated from different healthy individuals are shown. Size bars 20 µm. <b>B.</b> MCF-10A cells stained for Ki-67 (green) and actin (red) after 3, 6, 12, and 21 days of 3D-culture in ECM-derived substrate under normoxic (21%) or hypoxic (1%) conditions. Representative images from one of three independent experiments are shown. Size bars 20 µm. <b>C.</b> Percentage of cells with Ki-67 positive nuclei in normoxic and hypoxic MCF-10A cell organoids 3, 12, and 21 days post-seeding, in three independent experiments. Statistical analysis was performed with Student’s paired t-test (p). In each experiment at least 200 cells were included in the calculation. <b>D.</b> Cell death in MCF-10A cells grown in 3D-culture under normoxic and hypoxic conditions for 12 days, by <i>in situ</i> cell death detection (red), nuclear staining with DAPI (blue). All confocal micrographs were acquired at the Z-plane where the depictured acini-like structure had the widest circumference. Size bars 20 µm. <b>E.</b> Percentage of cells with nuclei positive for <i>in situ</i> cell death detection in normoxic (21%) and hypoxic (1%) 3D-cultures at 9 and 12 days post-seeding. Data from four experiments are shown. In each experiment at least 200 cells were included in the calculation.</p

Loss of polarization in hypoxic breast epithelial cells.

<p><b>A.</b> Positive HIF-1α IHC staining of hypoxic cells (broken arrow) adjacent to the necrotic zone (star) in ductal carcinoma <i>in situ</i> of the breast. Small duct-like formations (arrows) in non-hypoxic regions close to the basal membrane in two different patient specimens of ductal carcinoma <i>in situ</i>. H/E; haematoxylin/eosin staining. Size bars 20 µm. <b>B</b>. Size and polarization of human breast epithelial cell acini grown on ECM-derived substrate at 21% and 1% oxygen. Actin (phalloidin, red) and nuclear (DAPI, blue) staining of normoxic (upper panel) and hypoxic (lower panel) primary human breast epithelial cells (left panels) and MCF-10A cells (right panel) at the indicated days post-seeding. The primary breast epithelial cell micrographs are from one representative time-series out of three sets of cultured breast cell samples from three different healthy women. All confocal micrographs were acquired at the Z-plane where the depictured acini-like structure had the widest circumference. Size bars 20 µm. <b>C.</b> Number of MCF-10A cell organoids of a given diameter (left) and the average size (right) of MCF-10A cell organoids cultured at 21% or 1% oxygen for 21 days on ECM-derived substrate. <b>D.</b> Number of polarized MCF-10A cell organoids of the given diameter (left) and the percentage of polarized organoids (right) after 21 days of culture on ECM-derived substrate at 21% or 1% oxygen. Data from one representative experiment out of three is shown. Organoids were considered polarized if 50% or more of the cells in the outer layer formed a palisade.</p